Metagenomic Analysis of Sputum Samples from Cystic Fibrosis Patients
Cystic Fibrosis Cystic fibrosis is an autosomal recessive genetic disorder. It is defined as a disease of the secretory glands of the body, namely the sweat and mucosal glands. In the average human body, mucous is normally slippery and wet, providing lubrication for many bodily functions. In the body of a CF patient, mucous is thick, sticky, and dehydrated. The symptoms and severity of CF varies patient to patient, but most experience debilitating lung and digestive problems. Many CF patients have very salty sweat, indicating they are losing a large amount of salt from the body. Respiratory problems are common and are related to a build up of mucous in the airways. The build up of mucous inhibits the normal function of certain organs, namely the pancreas, preventing digestive enzymes from reaching the intestine. Patients with CF are at a higher risk for developing diabetes, osteoporosis, and fertility issues. The most common cause of death in CF patients is respiratory failure related to mucous build up. While the predicted lifespan of someone with CF is increasing, most patients do not live past their 20s or 30s. Therapies for CF include specific diets, medicines, exercise, and respiratory therapies (1). The Goals of this Metagenomic Analysis Researchers have been interested in a metagenomic analysis of the bacteria, viruses, and fungi associated with the lungs of CF patients in the hopes of one day developing personalized diagnoses and treatments. The infections that CF patients encounter are nearly always polymicrobic and require multiple rounds of treatments; by determining the microbes most commonly encountered (as well as antibiotic resistance genes encountered), broad-spectrum treatment practices can be narrowed down to serve patients on an individual basis (2). The goals of this particular metagenomic analysis include (2): * Discovering the total community DNA from sputum samples * Discovering any and all antibiotic resistance genes * Discovering any microbial metabolic pathways * Tracking how antibiotic resistance in microbes can fluctuate over time * Tracking how microbial communities can differ among areas in the lungs The Metagenomic Analysis The sputum samples of 3 adult patients with CF were collected and analyzed; each patient provided 10 samples apiece. Patients were included based on the following criteria (2): * A diagnosis of CF (including two known CFTR gene mutations) * An increase in respiratory symptoms associated with CF * A decrease in lung expiratory volume The sputum samples were collected at the following points in time (2): * Beginning of exacerbation period (before antibiotic therapy) * Within a 24 hour period of beginning antibiotic therapy * Within a 12 hour period of completing antibiotic therapy * 4 weeks after completing antibiotic therapy Samples were collected and aliquotted separately for distinct isolation of bacterial life and viral particles. Mucous aliquots for viral particle detection were: treated with DTT, passed through a micropore filter, isolated and concentrated, prepared for microbial DNA extraction. Mucous aliquots for bacterial detection were: treated with β-mercaptoethanol, separated from human cells, had extracellular DNA removed, prepared for microbial DNA extraction (2). Analysis Results Patient #1 - moderate lung disease * Fungal growth: Candida albicans; Scedosporium apiospermum. * Bacterial growth: extended spectrum β-lactamase (ESBL) Escherichia coli. * Additional growth: Streptococcus spp. (oral cavity contaminant); Rothia mucilaginosa; Pseudomonas aeruginosa (very low amounts). * E. coli was detected in high numbers before antibiotic therapy; numbers decreased throughout the entirety of the antibiotic therapy; numbers increased after therapy was completed. * Streptococcus spp. and R. mucilaginosa numbers increased throughout the course of antibiotic therapies (2). Patient #2 - severe end-stage lung disease * Fungal growth: Candida albicans; Aspergillus fumigatus. * Bacterial growth: Stenotrophomonas maltophilia; Pseudomonas aeruginosa; Rothia spp. '' * Additional growth: ''Streptococcus spp. (oral cavity contaminant). * P. aeruginosa, Streptococcus spp., and Rothia spp. were found in large amounts pre-antibiotic therapy; numbers decreased throughout the entirety of antibiotic therapy. * After therapy was completed, S. maltophilia ''was detected in high amounts and had adapted to be multi-drug resistant (2). Patient #3 - mild lung disease * Bacterial growth: ''Pseudomonas aeruginosa; Streptococcus ''group C; ''Stenotrophomonas maltophilia; Rothia ''spp. * Additional growth: ''Veillonella parvula ''(oral cavity contaminant); ''Fusobacterium nucleatum ''(oral cavity contaminant). * ''P. aeruginosa, R. mucilaginosa, and Lactobacillus ''spp. were all found in abundance after the completion of antibiotic therapy (2). Conclusions Researchers came to the conclusions that (2): # Each CF patient was housing a unique and diverse microbial environment. # Microbial communities in the lung of a CF patient differ across periods of time. # An alarmingly high number of antibiotic resistance genes were discovered (>50% of which were efflux pump-mediated mechanisms of resistance). References 1) What is Cystic Fibrosis? NIH. http://www.nhlbi.nih.gov/health/health-topics/topics/cf/. Updated December 26, 2013. Accessed November 9, 2014. 2) Lim YW, Evangelista JS 3rd, Schmeider R, et. al. Clinical insights from metagenomic analysis sputum samples from patients with cystic fibrosis. ''J Clin Microbiol. February 2014; 52(2): 425-437. doi: 10.1128/JCM.02204-13.